Method for high resolution zone electrophoresis

ABSTRACT

A method of separating particles by zone electrophoresis by applying a potential gradient in one direction, allowing particles to migrate and build-up against a barrier, then reversing the current to allow the particles to migrate away from the barrier.

United States Patent 1 Juhos n 1 3,720,593 1March 13, 1973 METHOD FORHIGH RESOLUTION ZONE ELECTROPHORESIS Inventor: Eva Th. ,Iuhos, MountainView,

Calif.

Assignee: Beckman Instruments, Inc.

Filed: Jan. 17, 1972 Appl. No.: 218,613

Related US. Application Data Division of Ser. No. 587,013, Oct. 17,1966, abandoned.

US. Cl. ..204/l80 G, 204/299 Int. Cl. ..B0lk 5/00 Field ofSearch....204/l80 P, 180 G, 180 R, 299

Primary Examiner-John H. Mack Assistant Examiner-A. C. PrescottAttorney-Thomas L. Peterson et al.

[57] ABSTRACT A method of separating particles by zone electrophoresisby applying a potential gradient in one direction, allowing particles tomigrate and build-up against a barrier, then reversing the current toallow the particles to migrate away from the barrier.

2 Claims, 2 Drawing Figures PATENTEDHAR 1 3197s EVA TH. JUHOS INVENTOR.

ATTORNEY METHOD FOR HIGH RESOLUTION ZONE ELECTROPHORESIS This is adivision of my copending application Ser. No. 587,013, filed Oct. 27,1966 now abandoned, entitled METHOD AND APPARATUS FOR HIGH RESOLUTIONZONE ELECTROPHORESIS, assigned to the assignee of the presentapplication.

This invention relates generally to zone electrophoresis and moreparticularly to a method and an apparatus for performing zoneelectrophoresis in which rapid, high resolution separations areobtained.

- Electrophoresis, in general, is the migration of particles in anelectrolytic carrier medium under the influence of an electric field.This phenomenon can be used to separate particles which are chemicallysimilar but which exhibit distinctive surface electrical properties. Asa result of these distinctive electrical properties, the mobilities ofvarious classes of charged particles in the carrier medium, under theinfluence of the electric field, will be different. Particles having thesame electrical propertiesmigrate together in specific, identifiablezones.

According to one method of separating'charged particles by zoneelectrophoresis, a sample containing the particles is appliedto anelectrolytic carrier medium in the form of a porous substance or agel-like matrix such large particles such as protein; small pores act asa partial or full barrier, slowing or completely stopping the migrationof larger particles. Particles whose migration velocity is the same willmigrate inspecific zones which may be analyzed quantitatively andqualitatively by methods well known in" the electrophoresis art. Thedirection of migration of a particular particlewill depend upon thepolarity of its surface charge.

One disadvantage of prior zone electrophoresis methods and apparatus inwhich a gel matrix is utilized, is that unless the initial sample iscarefully applied as a thin line, electrophoretic separation is slow andlow resolution zones are produced. The narrower and sharper the zone ofinitial sample application, the more rapidly separation occurs and themore highly resolved the resulting electrophoretic separations will be.Applying the initial sample as a thin, sharp line however, is difficultto accomplish and reproducibility is poor.

Accordingly, it is the overall object of the present invention to,provide a method and apparatus for accomplishing zone electrophoresisyielding rapid, high resolution separations. 4

In accordance with one specific, exemplary form of the present inventionshown and described herein, there is provided a novel methodandapparatus for practicing same, in which the sampleparticles'areinitially moved in the carrier medium by an electricalpotential gradient against a "barrier" through which ordinary ions willpass but which is impermeable to the particles of interest, theseparticles being of macromolecular size. The particles ofinterestconcentrate at the barrier in the form of a fine line or film whichprovides a sharply localized initial sample. The polarity of thepotential gradient is then reversed and the particles concentrated atthe barrier begin to migrate in a direction away from the barrier. Theelectrophoresis which now takes place results in sharply definedparticle zones susceptible of highly accurate analysis.

The novel features which are believed to be characteristic of theinvention are set forth with particularity in the appended claims. Theinvention itself, however, together with further objects and advantagesthereof can best be understood by reference to the following descriptiontaken in connection with the accompanying drawings in which:

FIG. 1 shows an elevation view, in section, ofan ex- 7 emplaryembodiment of an apparatus which may be used to practice thepresentinvention; and

FIG. 2 is a perspective view of another exemplary embodiment of anapparatus for practicing the invention.

Referring now to the drawings, there is shown in FIG. 1a zoneelectrophoresis device comprising generally a carrier medium supportmeans which may take the form of an open-ended tube 10. The tube 10 ispacked with an electrophoresis carrier medium 12 typically comprising agel such as agar, starch, acrylamide or the like. The tube may beoriented in any convenient direction; in the specific, exemplaryconfiguration depicted, the tube 10 is vertically disposed and supportedby means of a suitable clamping arrangement (not shown).

A barrier means is provided across the tube 10 to stop the migration ofparticles of interest while permitting the passage of ordinary ions. Inthe embodiment shown in FIG. 1, the barrier is in the form of a membrane14 stretchedacross the open top of the tube 10 and in contact with-thecarrier'medium inside. An interface l6 is thereby defined at-thejunction of the membrane 14 and the carrier medium 12. The membrane 14may be fabricated of any suitable material so long as it isimpermeable'to the particles of interest yet provides a conductive pathfor the electrical potential gradient applied to the carrier medium. Forthe separation of certain types of proteinaceous matter, cellophanehasbeen found to be a satisfactory barrier membrane.

' The barrier need not be in the form of a membrane, however. A firmgel, that is, one having small pores through which larger particles ofinterest cannot pass, may be utilized. The firm gel may be east alongwith the carrier medium (arelatively weak, or large pore gel) formingtwo. gel zones or portions defining an interface between. Y

The sample, containing the particles of interest which are tobeseparated, may be pre-mixed with the gel or introduced in other wellknown ways, as by ldiffusion, for example. In the apparatus of FIG. 1,the sample-containingv portion of the gel, denoted by the referencenumeral 18 is precast with the carrier medium 12 and islocated adjacentthe the upper end of the tube 10. The position of the sample along thelength of the tube is immaterial, except that it is desirable to havethe sample located initially near the barrier so that the time requiredfor the particles of interest to migrate to the barrier is minimized.

Electrode vessels 20 and 22, containing a buffer solution 23 with anappropriate pI-I, are provided adjacent the upper and lower ends of thetube 10. The vessel 20 may be conveniently fabricated of a length ofglass or plastic tubing. The vessel 20 is held in place over the upperend of the tube by a tapered, rubber support 24 having a central hole 25which fits snugly over the upper portion of the tube 10. The support 24may double as a clamp to hold the membrane 14 in place. With thearrangement shown, the buffer solution 23 in the vessel is in contactwith the upper surface of the membrane 14. The lower portion of the tube10 is immersed in the buffer solution 23 contained in vessel 22, thelower end of the gel in the tube 10 being thereby brought into contactwith the buffer.

The vessels 20 and 22 are provided with electrodes 26 and 28,respectively, and a dc potential from a battery 30 or other suitablesupply is applied between the electrodes. A means for selectivelyreversing the polarity of the electric field between the electrodes 26and 28 may also be provided. This may take the form of a double-pole,double-throw switch 32 connected to the electrodes 26 and 28 and to thesupply 30 in a well known fashion so that with the switch in oneposition the electrode 26 will be positive with respect to the electrode28 and with the switch 30 in the other position the polarity of theelectrodes will be reversed.

In operation, assuming a protein sample is utilized, the switch 32 ispositioned so that the upper electrode 26 acts as the anode. Theproteinaceous matter then moves up to and concentrates in a thin film atthe membrane barrier 14. The polarity of the electrodes 26 and 28 isthen reversed, causing the particles of interest to migrate downwardlyin the carrier medium undergoing electrophoretic separation into highlyresolved, distinct zones.

Turning now to FIG. 2, the alternative apparatus depicted thereinincludes an elongated, horizontally disposed, flat glass plate 50 forsupporting a carrier medium in the form of a suitable gel film 52. Theends of the film 52 are connected to the buffer solutions 54 in theelectrode vessels 56 and 58 by filter paper wicks 60 and 62,respectively. Electrodes 64 and 66 connect the buffer solution in theelectrode vessels 56 and 58 with a suitable d.c. supply 68 through apolarity reversing means such as the double-pole, double-throw switch70. A barrier, in the form of a membrane 72, as described in connectionwith FIG. 1, and supported by the plate 50, extends upwardly from theplate and across the film 52 thereby dividing the film into two portions74 and 76. The sample may be applied in any suitable manner to the filmportion 74. The operation of the device of FIG. 2 is essentially thesame as that described for FIG. 1, consisting generally inelectrophoretically moving the particles of interest toward the barrierfor concentration there followed by reversal of the polarity of thepotential gradient to effect particle separation.

Instead of employing a membrane barrier in the apparatus of FIG. 2, itis also feasible to form the film portion 76 of a firm gel, the filmportion or carrier medium 74 comprising a relatively weak gel. Theparticles will then concentrate at the interface defined by the two filmportions in the manner already described.

It will be obvious to those skilled in the art that variousmodifications may be made to the specific exemplary embodiments o theinvention described. While particular embodiments have been discussed,it will be understood that the invention is not limited thereto and thatis is contemplated to cover in the appended claims any suchmodifications as fall within the true spirit and scope of the invention.

What is claimed is:

l. A method for separating particles by zone electrophoresis, comprisingthe steps of applying to an electrolytic carrier medium a quantity ofsample including said particles, said medium having a barrierimpermeable to said particles; applying to said carrier medium apotential gradient in a first direction causing said particles tomigrate toward said barrier and concentrate at said barrier; removingsaid potential gradient; and

applying to said carrier medium a potential gradient in a seconddirection to cause said particles to migrate away from said barrier andundergo electrophoretic separation. 2. A method of obtaining rapid, highresolution electrophoretic separations of particles by electrophoresis,comprising the steps of applying to an electrolytic carrier medium aquantity of a sample including said particles, said medium having abarrier impermeable to said particles;

applying to said carrier medium a potential gradient the polarity ofwhich causes said particles to migrate toward said barrier andconcentrate in the form of a thin film at said barrier and reversing thepolarity of said potential gradient to cause said particles to migrateaway from said barrier and undergo electrophoretic separation in saidcarrier medium.

a: t 1: a:

1. A method for separating particles by zone electrophoresis, comprisingthe steps of applying to an electrolytic carrier medium a quantity ofsample including said particles, said medium having a barrierimpermeable to said particles; applying to said carrier medium apotential gradient in a first direction causing said particles tomigrate toward said barrier and concentrate at said barrier; removingsaid potential gradient; and applying to said carrier medium a potentialgradient in a second direction to cause said particles to migrate awayfrom said barrier and undergo electrophoretic separation.